The present invention relates to transgenic plant cells and plants with an increased activity of an amylosucrase protein and an increased activity of a branching enzyme. Such plant cells and plants synthesize a modified starch and/or synthesize xcex1-1,6 branched xcex1-1,4-glucans with a modified branching degree in O-6-position and/or give a higher yield in comparison with corresponding genetically non-modified wild type plants (plant cells).
In the area of agriculture and forestry it has been a permanent endeavor to produce plants with increased yield, in particular, in order to ensure the food for the continuously growing population of the world and to guarantee the supply of regenerating raw materials. Traditionally, attempts have been made to obtain productive plants by breeding. For each plant species of interest a corresponding breeding program has to be carried out. This is, however, time- and work-intensive. Progress has been made, partly by genetic manipulation of plants, i.e. by purposeful introduction and expression of recombinant nucleic acid molecules in plants. Such approaches have the advantage that, in general, they are not being limited to one plant species but can be transferred to other plant species. Therefore it seems desirable to provide plant cells and plants which give increased yields as well as to offer methods for the production of such plant cells and plants.
With regard to the growing importance which has been attached to vegetable substances as a source of regenerating raw material recently, it is one of the tasks in biotechnological research to strive towards adjusting these vegetable raw materials to the demands of the manufacturing industry. In order to facilitate the use of regenerating raw materials in as many application areas as possible it is furthermore essential to achieve a great variety of substances. Moreover, it is necessary to increase the yield of these vegetable substances in order to increase the efficiency of the production of sources of regenerating vegetable raw materials.
Apart from oils, fats and proteins, polysaccharides are the most important regenerating vegetable raw materials. Apart from cellulose, starch plays a vital role with the polysaccharides as it is one of the most important reserve substance in higher plants.
Apart from its use in foods, the polysaccharide starch is also widely used as regenerating raw material for the production of industrial products. The polysaccharide starch is composed of chemically uniform basic components, the glucose molecules, but forms a complex mixture of various molecules which have differing polymerization and branching degrees and therefore differ substantially in their physical and chemical properties.
A differentiation is made between amylose starch, a basically non-branched polymer composed of (xcex1-1,4-glycosidically linked glucose units, and the amylopectin starch, a branched polymer wherein branching is caused by the occurrence of additional xcex1-1,6-glycosidic links. According to the literature (Voet and Voet, Biochemistry, John Wiley and Sons, 1990) xcex1-1,6-glycosidic links occur on average at every 24th to every 30th glucose residue. This corresponds to a branching degree of about 3%-4%. Details of the branching degree are variable and depend on the source (e.g. plant species, plant variety, etc.) of the individual starch. Plants typically used for the industrial production of starch vary in their amylose content of the total starch content between 10 and 25%.
In order to facilitate a very wide use of polysaccharides such as e.g. starch it seems desirable to provide plants which are modified in their polysaccharide composition and, for example, are able to synthesize modified starch and/or highly branched xcex1-1,6-xcex1-1,4-glucans which are particularly suitable for various uses. One possibility to produce such plants isxe2x80x94apart form breeding methodsxe2x80x94the purposeful modification of the starch metabolism in starch producing plants by genetic engineering methods, A prerequisite hereto, however, is the identification and characterization of the enzymes playing a role in the starch synthesis and/or modification as well as the isolation of the corresponding DNA molecules encoding these enzymes. The biochemical synthesis pathways which lead to the formation of starch are essentially known. The starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissues these are the chloroplasts, in photosynthetically inactive starch-storing tissues the amyloplasts.
The most important enzymes participating in the starch synthesis are the starch synthases (cf. for example patent application WO 96/15248), the R1-enzyme (cf. for example WO 97/11188) as well as the branching enzymes (cf. for example WO 92/14827). The exact role of other enzymes such as e.g. the starch phosphorylases (cf. for example WO 98/40503) during starch biosynthesis is not known. In order to provide further possibilities to modify any plants in such a way that they synthesize modified starch, it is also possible to introduce foreign nucleic acid molecules, as e.g. bacterial or fungal, which are not present in wild type plants and which encode proteins participating in the synthesis of polysaccharides. It could be shown, for example, that the synthesis of so-called xe2x80x9cAmylofructanxe2x80x9d is possible by amyloplastidic expression of bacterial fructosyltransferases in amyloplasts (Smeekens, Trends in Plant Science Vol. 2 No. 8 (1997), 286-288).
The heterologous expression of a bacterial glycogen synthase in potato plants leads to a slight decrease in the amylose content, an increase of the branching degree and a change in the branching pattern of the amylopectin in comparison with wild type plants (Shewmaker et al., Plant. Physiol., 104 (1994), 1159-1166).
Moreover, the expression of a bacterial branching enzyme in potato plants in amylose-free potato mutants (amf) (Jacobsen et al., Euphytica, 44 (1989), 43-48) leads to amylopectin molecules having 25% more branching points (Kortstee et al., The Plant Journal, 10(1), (1996), 83-90) than the control molecules (amf). The increase in branching points was due to a modification of the distribution of the chain length of longer side chains in favor of shorter side chains. The reduction of the average chain-length and the reduction of the xcexmax after iodine staining also are an indication for a higher branched structure of the amylopectin in transformed plants in comparison with non-transformed plants (Kortstee et al., see above). The branching degree of glycogen of about 10% could, however, not be achieved via this approach. Glycogen, a polysaccharide, which is found mainly in animals and bacteria, contains highly-branched xcex1-1,6-xcex1-1,4-glucans. Glycogen differs from starch also in the average length of the side chains and in the polymerization degree. According to the literature (Voet and Voet, Biochemistry, John Wiley and Sons, 1990) it contains an xcex1-1,6-branching point at every 8th to 12th glucose residue on average. This corresponds to a branching degree of about 8% to 12%. There are various figures for the molecular weight of glycogen which vary between 1 million and more than 1000 millions (D. J. Manners in: Advances in Carbohydrate Chemistry, Ed. M. L. Wolfrom, Academic Press, New York (1957), 261-298; Geddes et al., Carbohydr. Res., 261(1994), 79-89). Theses figures, too, very much depend on the corresponding source organism, its nutritional state as well as the kind of isolation of glycogen. Usually it is obtained by costly and time-intensive methods from mussels (e.g. Mytillus edulis), from mammal livers or muscles (e.g. rabbits, rats) (Bell et al., Biochem. J. 28 (1934), 882; Bueding and Orrell, J. Biol. Chem., 236 (1961), 2854). Moreover, in plants one finds, for example, in the su1-mutant of maize the so-called phytoglycogen which has a branching degree of about 10% and which shows, in comparison with amylopectin a modified side chain distribution (Yun and Matheson, Carbohydrate Research 243, (1993), 307-321) and a different solubility behavior. Such phytoglycogen-accumulating plants, however, show a reduction in the starch content of up to 90% (Zeeman et al., Plant Cell 10, (1998), 1699-1711).
Furthermore, an in vitro-method using amylosucrase and a branching enzyme for the synthesis of xcex1-1,6-branched xcex1-1,4-glucans was described which amongst others allows for the production of highly-branched (glycogen-similar) glucans (German Patent Application DE 19846635.8). The production of such glucans in plants, however, is not described therein.
Therefore it seems desirable to provide alternative means which allow for the reasonably-priced production of modified starches and/or of xcex1-1,6-xcex1-1,4-glucans with a modified branching degree in O-6-position in comparison with wild type plants in plants.
Thus, the technical problem underlying the present invention is to provide plant cells and plants which, in comparison with corresponding non-modified wild type plant cells and wild type plants, contain a modified composition of the polysaccharides contained in the plant cells and plants and, if possible, also show a higher yield.
This problem has been solved by providing the embodiments characterised in the claims.
Therefore, the present invention relates to transgenic plant cells which are genetically modified wherein the genetic modification is the introduction of one foreign nucleic acid molecule or several foreign nucleic acid molecules the presence or the expression of which leads to an increased activity of an amylosucrase protein and an increased activity of a branching enzyme protein in comparison with corresponding genetically non-modified plant cells of wild type plants.
The genetic modification can be any genetic modification which leads to an increase in the amylosucrase activity and to an increase in the branching enzyme activity.
In a preferred embodiment the genetic modification consists of the introduction of one foreign nucleic acid molecule encoding an amylosucrase protein and a branching enzyme into the genome of a plant cell.
This foreign nucleic acid molecule can, for example, be a so-called xe2x80x9cdouble-constructxe2x80x9d which is a single vector for plant transformation which contains the genetic information encoding both for an amylosucrase protein and for a branching enzyme.
The nucleic acid molecules coding for the amylosucrase enzyme and for the branching enzyme which are both contained in the xe2x80x9cforeign nucleic acid moleculexe2x80x9d can either, independently from each other, be under control of a promoter each or they can, after fusion as translational unit, be under control of the same promoter.
In another preferred embodiment several foreign nucleic acid molecules are introduced into the genome of the plant cell wherein one foreign nucleic acid molecule encodes an amylosucrase protein and a further foreign nucleic acid molecule encodes a branching enzyme.
Hereby, the foreign nucleic acid molecules can be introduced into the genome of the plant cell at the same time or consecutively. In the first case it is called a xe2x80x9ccotransformationxe2x80x9d, in the latter a xe2x80x9csupertransformationxe2x80x9d.
The term xe2x80x9ctransgenicxe2x80x9d therefore means that the plant cell of the invention contains at least one foreign, preferably two foreign nucleic acid molecule(s) stably integrated in the genome, preferably one or two nucleic acid molecules encoding an amylosucrase protein and a branching enzyme.
The term xe2x80x9cforeign nucleic acid moleculexe2x80x9d preferably means a nucleic acid molecule encoding a protein with amylosucrase activity and a protein with branching enzyme activity and which either does not occur in the corresponding plants naturally or which does not occur naturally in the actual spatial order in the plants or which is located at a place in the genome of the plant where is does not occur naturally. Preferably, the foreign nucleic acid molecule is a recombinant molecule consisting of various elements the combination or the specific spatial order of which does not occur naturally in plants. The plants of the invention contain at least one foreign nucleic acid molecule encoding a protein with amylosucrase activity and a protein with branching enzyme activity preferably linked with regulatory DNA elements which guarantee the transcription in plants, in particular with a promoter.
The term xe2x80x9cseveral foreign nucleic acid moleculesxe2x80x9d preferably means two nucleic acid molecules wherein one foreign nucleic acid molecule encodes an amylosucrase protein and the second foreign nucleic acid molecule encodes a branching enzyme.
In principle, the foreign nucleic acid molecule(s) can be any nucleic acid molecule(s) coding for an amylosucrase protein and a branching enzyme.
Within the present invention an amylosucrase protein (sucrose:1,4-xcex1-D-glucan 4-xcex1-glucosyltransferase, E.C.2.4.1.4.) refers to an enzyme which, preferably in vitro, catalyses the conversion of sucrose into water-insoluble xcex1-1,4-glucans and fructose. The following reaction scheme is suggested for this enzyme:
Sucrose+(xcex1-1,4-D-glucan)nxe2x86x92D-fructose+(xcex1-1,4-D-glucan)n+1 
This is a transglycosylation reaction. The products of this in-vitro-reaction are water-insoluble xcex1-1,4-glucans and fructose.
Nucleotide-activated sugars or cofactors are not necessary for this reaction. The enzyme, however, is stimulated in vitro by the presence of glucosyl group acceptors (or primers), as e.g. maltooligo saccharides, dextrin or glycogen onto which the glucosyl residue of the sucrose is transferred according to the reaction scheme above with concomitant xcex1-1,4-glucan chain extension (Remaud-Simeon et al., in S. B. Petersen, B. Svenson and S. Pedersen (Eds.), Carbohydrate bioengineering, 313-320 (1995); Elsevier Science B. V., Amsterdam, Netherlands).
Within the present invention, in principle, all amylosucrases are suitable which catalyze the synthesis of linear xcex1-1,4-glucans from sucrose.
Amylosucrases have so far only been known from bacteria species, in particular mainly from the Neisseria-species (MacKenzie et al., Can. J. Microbiol. 24 (1978), 357-362).
Therefore an amylosucrase of procaryotic origin is used preferably. Amylosucrases are known, for example, from Neisseria perflava (Okada and Hehre, J. Biol. Chem. 249 (1974), 126-135; MacKenzie et al., Can. J. Microbiol. 23 (1977), 1303-1307) or Neisseria canis, Neisseria cinerea, Neisseria denitrificans, Neisseria sicca and Neisseria subflava (MacKenzie et al., Can. J. Microbiol. 24 (1978), 357-362). Furthermore, WO 95/31553 and PCT/EP 98/05573 describe an amylosucrase from Neisseria polysaccharea. 
In another preferred embodiment of the invention the foreign nucleic acid molecule encodes an amylosucrase from a bacterium of the genus Neisseria.
In a particularly preferred embodiment of the invention the foreign nucleic acid molecule encodes an amylosucrase from Neisseria polysaccharea, more preferably an amylosucrase with the nucleic acid or amino acid sequence as disclosed in the international patent application PCT/EP 98/05573.
The enzyme which is expressed in Neisseria polysaccharea is extremely stable, is attached firmly to the polymerization products and is competitively inhibited by the reaction product fructose (MacKenzie et al., Can. J. Microbiol. 23 (1977) 1303-1307). With the Neisseria-species Neisseria polysaccharea the amylosucrase is secreted (Riou et al., Can. J. Microbiol. 32 (1986), 909-911), whereas with other Neisseria-species it remains in the cell.
A branching enzyme (xcex1-1,4-glucan: xcex1-1,4-glucan 6-glycosyltransferase, E.C. 2,4.1.18) is a protein catalyzing a transglycosylation reaction wherein xcex1-1,4-links of an xcex1-1,4-glucan donor are hydrolyzed and the xcex1-1,4-glucan chains set free in this process are transferred onto an xcex1-1,4-glucan acceptor chain and thereby transformed into xcex1-1,6-links.
In connection with the present invention, in principle, all branching enzymes of any origin (bacterial, fungal, plant, animal) are suitable, for example, branching enzymes from maize (see e.g. Baba et al., Biochem. Biophys. Res. Cormmun. 181 (1991), 87-94; Genbank Acc. No. AF072724, AF072725), from potato (Kossmann et al., Mol. Gen. Genet. 203 (1991), 237-244; Jobling et al., Genbank Acc. No. AJ011885), from rice (Mizuno et al., J. Biochem. 112 (1992), 643-651; Kawasaki et al., Mol Gen. Genet. 237 (1993), 10-16; Mizuno et al., J. Biol. Chem. 268 (1993), 190844-19091; Nakamura and Yamanouchi, Plant Physiol. 99 (1992), 1265-1266), from wheat (Baga et al., Plant Mol. Biol. 40 (1999), 1019-1030; Rahman et al,. Theor. Appl. Genet. 98 (1999), 156-163 and Genbank Acc. No. Y12320), from barley (Genbank Acc. No. AF064561), from Synechocystis (Genbank Acc. No. D63999), from E. coli (Baecker et al., J. Biol. Chem. 261 (1986), 8738-8743; Genbank Acc. No. M13751), from Bacillus stearothermophilus (Genbank Acc. No M35089), Streptomyces aureofaciens (Genbank Acc. No. L11647), Bacillus caldolyticus (Genbank Acc. No. Z14057), Synechococcus PCC6301 (Genbank Acc. No. M31544), Synechococcus sp. PCC7942 (Kiel et al., Gene 78 (1989), 9-17) and from Agrobacterium tumefaciens (Genbank Acc. No. AF033856).
The isolation of corresponding genes is possible for the person skilled in the art by means of molecular biological standard procedures, as described i.a. by Sambrook et al. (Sambrook et al., Molecular cloning; A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York, USA (1989)).
In a preferred embodiment of the invention the foreign nucleic acid molecule codes for a branching enzyme from a prokaryote, preferably from a bacterium of the genus Neisseria, particularly preferred from Neisseria denitrificans and even more preferred for a branching enzyme with the nucleotide sequence depicted in SEQ ID No.1 or with the amino acid sequence depicted in SEQ ID No. 2.
In a further preferred embodiment the foreign nucleic acid molecule codes for a plant branching enzyme.
There is a variety of techniques for the introduction of DNA into a plant host cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA with the biolistic approach as well as further possibilities.
The use of the Agrobacterium-mediated transformation of plant cells was examined intensively and was described sufficiently in EP 0 120516; Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and An et al. EMBO J. 4, (1985), 277-287. For the transformation of potato, see e.g. Rocha-Sosa et al., EMSO J. 8, (1989), 29-33).
The transformation of monocotyledonous plants by means of Agrobacterium-based vectors was described (Chan et al., Plant Mal. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271-282; Deng et al., Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11, (1992), 76-80; May et al., Bio/Technology 13, (1995), 486-492; Connor and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Trangenic Res. 2, (1993), 252-265). An alternative system for the transformation of monocotyledonous plants is the transformation with the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994). 37-4B; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625-631), the protoplast transformation, the electroporation of partially permeabilized cells, the introduction of DNA via glass-fibres. The transformation of maize, in particular, has been described in the literature repeatedly (cf. e.g. WO 95/06128, EP 0513849, EP 0465875, EP 0 292435; Fromm et al., Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-818; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726).
The successful transformation of other species of grain, too, has already been described, e.g. for barley (Wan and Lemaux, see above; Ritala et al., see above; Krens et al., Nature 296, (1982), 72-74) and for wheat (Nehra et al., Plant J. 5, (1994), 285-297).
In general any promoter active in plant cells can be used for the expression of the foreign nucleic acid molecule (of the foreign nucleic acid molecules). The promoter can be chosen in such a way that the expression in the plants of the invention occurs constitutively or only in a certain tissue, at a certain point in time of the development of the plant or at a time determined by external influential factors. With regard to the plant the promoter can be homologous or heterologous.
Appropriate promoters are e.g. the promoter of the 35S RNA of the Cauliflower Mosaic Virus and the ubiquitin promoter of maize for a constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for a tuber-specific expression in potatoes or a promoter which guarantees an expression only in photosynthetically active tissue, e.g. the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), the Ca/b promoter (see for example U.S. Pat. No. 5,656,496, U.S. Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89, (1992), 3654-3658) and the Rubisco SSU promoter (see for example U.S. Pat. No. 5,034,322, U.S. Pat. No. 4,962,028) or the glutelin promoter for an endosperm-specific expression (Leisy et al., Plant Mol. Biol. 14, (1990), 41-50; Zheng et al., Plant J. 4, (1993), 357-366; Yoshihara et al., FEBS Lett. 383, (1996), 213-218), the shrunken-1 promoter (Werr et al., EMBO J. 4, (1985). 1373-1380), the HMG promoter of wheat, the USP promoter, the phaseolin promoter or promoters of zein genes of maize (Pedersen et al., Cell 29, (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).
The expression of the foreign nucleic acid molecule (the foreign nucleic acid molecules) is particularly advantageous in those organs of the plant which have an increased sucrose content or which store sucrose. Such organs are e.g. the turnip of the sugar beet or the stem of sugar cane or of sugar millet. Therefore preferably used promoters are those which mediate the expression in these organs. Other promoters, however, can also be used, i.e. those which are only active at a point in time determined by external influential factors (cf. for example WO 9307279). Here, promoters of heat-shock proteins can be of special interest as they allow a simple induction. Furthermore, seed-specific promoters such as e.g. the USP promoter from Vicia faba, which guarantees a seed-specific expression in Vicia faba and other plants, can be used (Fiedler et al., Plant Mol. Biol. 22, (1993), 669-679; Bxc3xa4umlein et al., Mol. Gen. Genet. 225, (1991), 459-467). Moreover, fruit-specific promoters can be used, as described e.g. in WO 91/01373, WO 99/16879, and in van Haaren and Houck (Plant Mol. Biol. 21 (1993), 625-640).
In addition, a termination sequence can be present which is useful for the correct termination of transcription as well as for the addition of a poly-A-tail to the transcript which is ascribed a function in the stabilization of the transcripts. Such elements have been described in the literature (cf. e.g. Gielen et al., EMBO J. 8 (1989), 23-29 and are exchangeable arbitrarily.
The plant cells of the invention can be differentiated from naturally occurring plant cells inter alia by the fact that they contain one or more foreign nucleic acid molecule(s) which do(es) not naturally occur in these cells or that such (a) molecule(s) is (are) found integrated in such a place in the genome of the plants where it (they) do(es) not occur normally, i.e. in another genomic surrounding. Furthermore, such transgenic plant cells of the invention can be differentiated from naturally occurring plant cells as they contain at least one copy of the foreign nucleic acid molecule (foreign nucleic acid molecules) stably integrated in their genome, possibly in addition to copies of such a molecule which occur naturally in the plant cells. If the nucleic acid molecule(s) which is (are) introduced in the cell is (are) an additional copy (copies) of molecules occurring naturally in the plants then the plant cells of the invention can be differentiated from naturally occurring plant cells particularly by the fact that this (these) additional copy (copies) is (are) located in places in the genome where it (they) do not occur naturally. This can be tested, for example, by Southern Blot analysis.
Moreover, the plant cells of the invention can be differentiated from naturally occurring plant cells preferably by one of the following features: if the introduced nucleic acid molecule(s) is (are) heterologous with regard to the plant, the transgenic plant cells show transcripts of the introduced nucleic acid molecules. These can be detected, for example, in the Northern Blot analysis. Preferably, the plant cells of the invention contain proteins which are encoded by the introduced foreign nucleic acid molecule(s). This can be tested, for example, by immunological methods, in particular by Western Blot analysis.
If the introduced molecule is homologous with regard to the plant, the transgenic plant cells of the invention can be differentiated from naturally occurring plant cells, for example, due to the additional expression of the introduced foreign nucleic acid molecules. The transgenic plant cells preferably contain more transcripts of the foreign nucleic acid molecules. This can be tested, for example, by Northern Blot analysis.
The term xe2x80x9cgenetically modifiedxe2x80x9d means that the plant cell is modified in its genetic information by introduction of one foreign nucleic acid molecule or several foreign nucleic acid molecules and that the presence or the expression of the foreign nucleic acid molecule(s) leads to a phenotypic change. Thereby phenotypic change preferably means a measurable change of one or more functions of the plants (plant cells). The plant cells of the invention, for example, show an increased activity of a protein with amylosucrase activity and of a protein with branching enzyme activity due to the presence or the expression of the introduced nucleic acid molecule.
In the frame of the present invention the term xe2x80x9cincreased activityxe2x80x9d means an increased expression of the nucleic acid molecule (several nucleic acid molecules) coding for a protein with amylosucrase activity and for a protein with branching enzyme activity, an increase in the amount of proteins with amylosucrase activity and with branching enzyme activity or an increase in the activity of a protein with amylosucrase activity and of a protein with branching enzyme activity in the plants.
An increase of the expression can be determined, for example, by measuring the amount of transcripts coding such proteins, e.g. by Northern Blot analysis. There, an increase preferably means an increase in the amount of transcripts in comparison with corresponding genetically non-modified plant cells by at least 10%, preferably by at least 20%, particularly preferred by at least 50% and especially preferred by at lest 75%.
The increase in the amount of protein with amylosucrase activity or with branching enzyme activity can be determined, for example, by Western Blot analysis. There, an increase preferably means an increase in the amount of protein with amylosucrase activity or with branching enzyme activity and/or an increase in the amylosucrase activity or the branching enzyme activity in comparison with corresponding genetically non-modified cells by at least 10%, preferably by at least 20%, particularly preferred by at least 50% and especially preferred by at least 75%.
The activity of the amylosucrase protein and the branching enzyme can, for example, be tested as described in the examples. Furthermore, the activity of a branching enzyme can be determined as described in Lloyd et al. (Biochem. J. 338 (1999), 515-521). The amylosucrase activity can also be determined as described below in the section xe2x80x9cMaterials and Methods . . . xe2x80x9d, section 3.
Surprisingly, it was found out, that plants containing such plant cells with increased activity of an amylosucrase and of a branching enzyme synthesize xcex1-1,6 branched xcex1-1,4-glucans with a modified branching degree in O-6-position which are not synthesized by corresponding genetically non-modified wild type plant cells. In one embodiment of the invention the plant cells of the invention contain xcex1-1,6-branched xcex1-1,4-glucans with a branching degree in O-6-position of at least 2%, preferably of at least 4%. In another embodiment the branching degree is at least 6%, preferably at least 8%, particularly preferred at least 10% and especially preferred at least 12%.
Within the frame of the present invention xe2x80x9cbranching degreexe2x80x9d means the average number of branches in O-6-position in comparison with all glucose units linked in a different way.
The branching degree can be determined via a methylation analysis, as, for example, described further below. General information about this method can also be found, for example, in xe2x80x9cAnalysis of Carbohydrates by GLC and MSxe2x80x9d (Biermann, C. J. and McGinnis, G. D. (eds.) CRC Press (1989), Chapter 9 by Carpita, N. C. and Shea, E. M., 157-216) or in Bjxc3x6rndal H. et al. (Angew. Chem., 82, (1970), 643-662; Int. Ed. Engl. 9, (1970), 610-619).
In another embodiment of the invention the plant cells of the invention synthesize modified starches which differ from starches of corresponding wild type plant cells in their physico-chemical properties, in particular the amylose/amylopectin ratio, the branching degree, the average chain length, the phosphate content, the pasting properties, the size and/or the form of the starch granule. In particular, such a starch can be modified with regard to viscosity and/or the gel forming ability of starch pastes in comparison with wild type starch.
In a further embodiment of the invention plants which contain the plant cells of the invention have a higher yield in comparison with corresponding genetcally non-modified wild type plants.
Within the present invention, the term xe2x80x9cwild type plantxe2x80x9d means that the plants served as starting material for the production of the plants of the invention, i.e. whose genetic information, apart form the introduced genetic modification, corresponds to that of a plant of the invention.
Here, the term xe2x80x9cincreased yieldxe2x80x9d means an increase of the yield by at least 5%, preferably by at least 10%, particularly preferred by at least 20% and especially preferred by at least 30%. The term xe2x80x9cincreased yieldxe2x80x9d means preferably an increase in the production of substances and/or biomass, in particular when measured based on the fresh weight per plant.
Such an increase in yield preferably relates to parts of plants which can be harvested such as seeds, fruit, storage roots, roots, tubers, blossoms, buds, shoots, stems or wood.
In accordance with the invention the increase in yield is at least 3% referring to the biomass and/or content substances in comparison with corresponding non-transformed plants of the same genome type if cultivated under the same conditions, preferably at least 10%, particularly preferred at least 20% and especially preferred at least 30% or even 40% in comparison with wildtype plants.
In a further embodiment of the present invention the plant cells of the invention have an increased caloric value in comparison with corresponding genetically non-modified wildtype plant cells.
The term xe2x80x9ccaloric valuexe2x80x9d is defined as the amount of energy (given in calories or joule) the body gets with the digestion of food and which is used to cover energy needs. The term xe2x80x9cincreased caloric valuexe2x80x9d means an increase in the calorific value by at least 5%, preferably by at least 10%, particularly preferred by at least 20% and especially preferred by at least 30%.
Plants with high caloric values are of interest to the food industry, in particular for the diet of people with high energy need, such as e.g. ill or older people, of infants or of competitive athletes.
In a preferred embodiment the nucleotide sequence encoding an amylosucrase enzyme and a branching enzyme comprise a protein targeting signal sequence which ensures localization in a specific cellular compartment, such as the vacuole or the plastids. In a particularly preferred embodiment the nucleotide sequences coding for the two enzymes comprise a protein targeting signal sequence ensuring that both enzymes are located in the same cellular compartment. In this context, the foreign nucleic acid molecule may comprise one or more protein targeting signal sequence(s) ensuring localization of the amylosucrase enzyme and the branching enzyme in the same cellular compartment. It is in particular possible that each coding region coding for the amylosucrase or the branching enzyme comprise more than one signal sequence or a combination of different signal sequences.
In a further embodiment of the invention the foreign nucleic acid molecule has one or more protein targeting signal sequence(s) mediating a vacuolar localization of the amylosucrase protein and of the branching enzyme.
The nucleic acid molecules coding for the amyosucrase enzyme and for the branching enzyme which are both contained in the xe2x80x9cforeign nucleic acid moleculexe2x80x9d can either be under control of one or of several protein targeting signal sequence(s) independently from each other or they can be under control of one or of several protein targeting signal sequence(s) together after fusion as translational unit.
In another embodiment of the invention the foreign nucleic acid molecules have one each or several protein targeting signal sequence(s) each mediating a vacuolar localization of the amylosucrase protein and the branching enzyme.
In this embodiment several foreign nucleic acid molecules are introduced into the genome of the plant cell wherein one foreign nucleic acid molecule encodes an amylosucrase protein and a further nucleic acid molecule encodes a branching enzyme. As mentioned earlier, the foreign nucleic acid molecules can be introduced into the genome of the plant cell simultaneously or consecutively.
Each of the foreign nucleic acid molecules contains one or more protein targeting signal sequence(s) mediating a vacuolar localization of each the amylosucrase protein and the branching enzyme wherein the protein targeting signal sequences can be identical or can be different from each other.
The N-terminal sequence (146 amino acids) of the patatin protein, for example, can be used as a vacuolar targeting sequence (Sonnewald et al., Plant J. 1, (1998), 95-106). In a preferred embodiment the signal sequence described in SEQ ID No.7 is used. Furthermore, the following signal sequences can be used as vacuolar targeting sequences: the N-terminal signal sequence of the acid invertase of tomato (Genbank Acc. No. LM81081) or of potato (Genbank Acc. No. L29099), the N-terminal signal sequence of the sporamin of sweet potato (Koide et al., Plant Physiol. 114 (1997), 863-870), the N-terminal signal sequence of the aleurain of barley (Vitale and Raikhel, Trends in Plant Science 4 (1999), 149-155), the N-terminal signal sequence of the proteinase inhibitor of potato (Genbank Acc. No. X04118) in combination with the C-terminal vacuolar targeting signal peptide of barley lectin (Vitale and Raikhel, loc. cit.).
Further vacuolar signal sequences are described for example by Matsuoka and Neuhaus, Journal of Experimental Botany 50, (1999), 165-174; Chrispeels and Raikhel, Cell 68, (1992), 613-616; Matsuoka and Nakamura, Proc. Natl. Acad. Sci. USA 88, (1991), 834-838; Bednarek and Raikhel, Plant Cell 3, (1991), 1195-1206; Nakamura and Matsuoka, Plant Phys. 101, (1993), 1-5. In general, a combination may be used comprising an N-terminal signal sequence, which ensures the transport of the respective protein into the endoplasmic reticulum, and a C-terminal vacuolar targeting sequence. An overview over vacuolar targeting sequences can be found in Chrispeels and Raikhel (Cell 68 (1992), 613-616).
Since the vacuole can usually store great amounts of sucrose which serves as substrate for the amylosucrase, this compartment is suitable to produce plant cells which, due to an increased activity of an amylosucrase protein and an increased activity of a branching enzyme synthesize xcex1-1,6-branched xcex1-1,4-glucans in the vacuole. In one embodiment of the invention these glucans in O-6-position have a branching degree of at least 1%, preferably of at least 4%, particularly preferred of at least 7% and especially preferred of at least 10%.
In a further embodiment of the invention the branching degree in O-6-position can be controlled by selecting transgenic plants showing different ratios of branching enzyme activity to amylosucrase activity.
In a particularly preferred embodiment plant cells according to the invention in which both, the amylosucrase and the branching enzyme are located in the vacuole, show an increased caloric value. For the definition of this term, see above.
In a further embodiment of the invention the foreign nucleic acid molecule has one or more protein targeting signal sequence(s) mediating a plastidic localization of the amylosucrase protein and the branching enzyme protein.
The nucleic acid molecules coding for the amylosucrase enzyme and for the branching enzyme which are both contained in the xe2x80x9cforeign nucleic acid moleculexe2x80x9d can either, independently from each other, be under control of one or more protein targeting signal sequence(s) each or they can, after fusion as translational unit, be under control of one or more protein targeting signal sequence(s).
In another embodiment of the invention the foreign nucleic acid molecules have one or more protein targeting signal sequence(s) each which mediates (mediate) a plastidic localization of the amylosucrase protein and of the branching enzyme protein.
In this embodiment several foreign nucleic acid molecules are introduced into the genome of the plant cell wherein one foreign nucleic acid molecule encodes an amylosucrase protein and a further foreign nucleic acid molecule encodes a branching enzyme. As mentioned earlier, the foreign nucleic acid molecules can be introduced into the genome of the plant cell simultaneously or consecutively.
Each of the introduced foreign nucleic acid molecules contains one or more protein targeting signal sequence(s) mediating a plastidic localization of each the amylosucrase protein and the branching enzyme protein wherein the protein targeting signal sequences are identical or different to each other.
The signal sequence of ferrodoxin:NADP+ oxidoreductase (FNR) from spinach, for example, can be used as signal sequence. The sequence contains the 5xe2x80x2 non-translated region as well as the flanking transit peptide sequence of the cDNA of the plastidic protein ferrodoxin:NADP+ oxidoreductase from spinach (nucleotide xe2x88x92171 to +165; Jansen et al., Current Genetics 13, (1988), 517-522),
In addition, for example, the transit peptide of the waxy protein from maize plus the first 34 amino acids of the mature waxy protein (Klxc3x6sgen et al., Mol. Gen. Get. 217, (1989), 155-161) can be used as signal sequence.
Other plastidic targeting sequences that can be used are: the signal sequence of the Rubisco small subunit (Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988). 846-850, Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994), 12760-12764), the signal sequence of the NADP-malate dehydrogenase (Gallardo et al., Planta 197 (1995), 324-332) and the signal sequence of the glutathion reductase (Creissen et al., Plant J. 8 (1995), 167-175).
In a preferred embodiment of the invention the transit peptide of the waxy protein of maize (see above) is used (see Example 1) without the first 34 amino acids of the mature waxy protein.
In a particularly preferred embodiment the plastidic signal sequence of the R1 protein from potato is used (Lorberth et al., Nature Biotechnology 16 (1998), 473-477).
With the amyloplastidic expression of bacterial fructosyltransferases it could be demonstrated that the plastids also contain sucrose which can be transformed into xe2x80x9camylofructanexe2x80x9d by the fructosyltransferases in amyloplasts (Smeekens, Trends in Plant Science Vol. 2 No. 8, (1997), 286-288). Therefore that compartment is also suitable for the combined expression of an amylosucrase gene and a branching enzyme gene and allows for the synthesis of modified starch which is modified, for example, in its physio-chemical properties, particularly the amylose/amylopectin ratio, the branching degree, the average chain length, the phosphate content, the pasting properties, the size and/or the form of the starch granule in comparison with starch synthesized in wildtype plants.
Therefore, in a further embodiment of the invention the transgenic plant cells of the invention synthesize modified starches.
In a preferred embodiment of the invention the gel stability of these starches is changed compared to starches extracted from wildtype plants. In a particularly preferred embodiment the maximal gel stability is increased by at least 20%, more preferably by at least 50%, even more preferably by at least 100% and especially preferred by at least 200% compared to starches extracted from wildtype plants. The gel stability can be determined as described in Example 9.
The starches isolated from the plant cells of the invention can also be modified according to methods known to the person skilled in the art and are suitable both in unmodified or modified form for various applications in the foods and non-foods sectors.
In principle, the application area of the starch can be subdivided into two big areas. One area comprises the hydrolysis products of starch, mainly glucose and glucan components which are obtained via enzymatic or chemical methods. They serve as starting material for further chemical modifications and processes, such as fermentation. With regard to reduction of costs the simplicity and the cost-efficient conduction of a hydrolysis method can be of importance. At present, it is mainly enzymatic when amyloglucoseidase is used. It would be conceivable to save costs by reducing the amount of enzymes used. A modification of the structure of the starch, e.g. surface extension of the grain, easier digestibility through a lower branching degree or a steric structure which limits the accessibility for the used enzymes could achieve that.
The other area wherein the starch is used due to its polymer structure as so-called native starch can be divided into two further areas of application:
Starch is a classic additive for various food, where essentially it serves the purpose of binding aqueous additives or causes increased viscosity or increased gel formation. Important characteristics are flowing and sorption behavior, swelling and pasting temperature, viscosity and thickening performance, solubility of the starch, transparency and paste structure, heat, shear and acid resistance, tendency to retrogradation, capability of film formation, resistance to freezing/thawing, digestibility as well as the capability of complex formation with e.g. inorganic or organic ions.
In this vast area starch can be used as an adjuvant in various production processes or as an additive in technical products. The main field where starch is used as an adjuvant is the paper and cardboard industry. In this field, starch is mainly used for retention (holding back solids), for sizing filler and fine particles, as solidifying substance and for dehydration. In addition, the advantageous properties of starch with regard to stiffness, hardness, sound, grip, gloss, smoothness, tear strength as well as the surfaces are made use of.
2.1 Paper and Cardboard Industry
Within the paper production process, a differentiation can be made between four fields of application, namely surface, coating, mass and spraying. The requirements on starch with regard to surface treatment are essentially a high degree of brightness, corresponding viscosity, high viscosity stability, good film formation as well as little formation of dust. When used in coating the solid content, a corresponding viscosity, a high capability to bind as well as a high pigment affinity play an important role. As an additive to the mass rapid, uniform, free-of-loss dispersion, high mechanical stability and complete retention in the paper pulp are of importance. When using the starch in spraying, corresponding content of solids, high viscosity as well as high capability to bind are also significant.
2.2 Adhesive Industry
A major field of application is, for instance, in the adhesive industry, where the starch is used in four areas: the use as pure starch glue, the use in starch glues prepared with special chemicals, the use of starch as an additive to synthetic resins and polymer dispersions as well as the use of starches as extenders for synthetic adhesives. 90% of all starch-based adhesives are used in the production of corrugated board, paper sacks and bags, composite materials for paper and aluminum, boxes and wetting glue for envelopes, stamps, etc.
2.3 Textile and Textile Care Industry
Another possible use of starch as adjuvant and additive is in the production of textiles and textile care products. Within the textile industry, a differentiation can be made between the following four fields of application: the use of starch as a sizing agent, i.e. as an adjuvant for smoothing and strengthening the burring behavior for the protection against tensile forces active in weaving as well as for the increase of wear resistance during weaving, as an agent for textile improvement mainly after quality-deteriorating pretreatment, such as bleaching, dying, etc., as thickener in the production of dye pastes for the prevention of dye diffusion and as an additive for warping agents for sewing yarns.
2.4 Building Industry
The fourth area of application of starch is its use as an additive in building materials. One example is the production of gypsum plaster boards, in which the starch mixed in the thin plaster pastifies with the water, diffuses at the surface of the gypsum board and thus binds the cardboard to the board. Other fields of application are admixing it to plaster and mineral fibers. In ready-mixed concrete, starch may be used for the deceleration of the sizing process.
2.5 Ground Stabilization
Furthermore, starch is advantageous for the production of means for ground stabilization used for the temporary protection of ground particles against water in artificial earth shifting. According to state-of-the-art knowledge, combination products consisting of starch and polymer emulsions can be considered to have the same erosion- and encrustation-reducing effect as the products used so far; however, they are considerably less expensive.
2.6 Use of Starch in Plant Protectives and Fertilizers
Another field of application is the use of starch in plant protectives for the modification of the specific properties of these preparations. For instance, starches are used for improving the wetting of plant protectives and fertilizers, for the dosed release of the active ingredients, for the conversion of liquid, volatile and/or odorous active ingredients into microcristalline, stable, deformable substances, for mixing incompatible compositions and for the prolongation of the duration of effect due to slower decomposition.
2.7 Drugs, Medicine and Cosmetics Industry
Starch may also be used in the fields of drugs, medicine and in the cosmetics industry. In the pharmaceutical industry, the starch may be used as a binder for tablets or for the dilution of the binder in capsules. Furthermore, starch is suitable as disintegrant for tablets since, upon swallowing, it absorbs fluid and after a short time it swells so much that the active ingredient is released. For qualitative reasons, medical lubricating powders and medical powders for wounds are based on starch. In the field of cosmetics, starch is used, for example, as carrier of powder additives, such as scents and salicylic acid. A relatively vast field of application for starch is toothpaste.
2.8 Starch as an Additive in Coal and Briquettes
Starch can also be used as an additive in coal and briquettes. By adding starch, coal can be quantitatively agglomerated and/or briquetted in high quality, thus preventing premature disintegrating of the briquettes. Barbecue coal contains between 4 and 6% added starch, calorated coal between 0.1 and 0.5%. Furthermore, starch becomes more and more important as a binding agent since adding it to coal and briquette can considerably reduce the emission of toxic substances.
2.9 Processing of Ore and Coal Slurry
Furthermore, the starch may be used as a flocculating agent in the processing of ore and coal slurry.
2.10 Starch as an Additive in Casting
Another field of application is the use of starch as an additive to process materials in casting. For various casting processes cores produced from sands mixed with binding agents are needed. Nowadays, the most commonly used binding agent is bentonite mixed with modified starches, mostly swelling starches.
The purpose of adding starch is increased flow resistance as well as improved binding strength. Moreover, swelling starches may fulfill other prerequisites for the production process, such as dispersability in cold water, rehydratisability, good mixability in sand and high capability of binding water.
2.11 Use of Starch Rubber Industry
In the rubber industry starch may be used for improving the technical and optical quality. Reasons for this are improved surface gloss, grip and appearance. For this purpose, starch is dispersed on the sticky gummed surfaces of rubber substances before the cold vulcanization. It may also be used for improving printability of rubber.
2.12 Production of Leather Substitutes
Another field of application for the modified starch is the production of leather substitutes.
2.13 Starch in Synthetic Polymers
In the plastics market the following fields of application are emerging: the integration of products derived from starch into the processing process (starch is only a filler, there is no direct bond between synthetic polymer and starch) or, alternatively, the integration of products derived from starch into the production of polymers (starch and polymer form a stable bond).
The use of the starch as a pure filler cannot compete with other substances such as talcum. That changes when the specific starch properties become effective and the property profile of the end products is thus clearly changed. One example is the use of starch products in the processing of thermoplastic materials, such as polyethylene. Thereby, starch and the synthetic polymer are combined in a ratio of 1:1 by means of coexpression to form a xe2x80x98master batchxe2x80x99, from which various products are produced by means of common techniques using granulated polyethylene. The integration of starch in polyethylene films may cause an increased substance permeability in hollow bodies, improved water vapor permeability, improved antistatic behavior, improved anti-block behavior as well as improved printability with aqueous dyes.
Another possibility is the use of the starch in polyurethane foams. Due to the adaptation of starch derivatives as well as due to the optimization of processing techniques, it is possible to specifically control the reaction between synthetic polymers and the starch""s hydroxy groups. The results are polyurethane films which get the following property profiles due to the use of starch: a reduced coefficient of thermal expansion, decreased shrinking behavior, improved pressure/tension behavior, increased water vapor permeability without a change in water acceptance, reduced flammability and cracking density, no drop off of inflammable parts, no halogen and reduced aging. Disadvantages that presently still exist are reduced pressure and impact strength.
Product development of film is not the only option any more. Also solid plastics products, such as pots, plates and bowls can be produced with starch content of more than 50%. Furthermore, the starch/polymer mixtures offer the advantage that they are biodegradable to a larger extent.
Furthermore, due to their extreme capability to bind water, starch graft polymers have gained utmost importance. These are products having a backbone of starch and a side lattice of a synthetic monomer grafted on according to the principle of radical chain mechanism. The starch graft polymers available nowadays are characterized by an improved binding and retaining capability of up to 1000 g water per g starch at a high viscosity. In the past few years these super absorbers have been more widely usedxe2x80x94mainly in the hygiene field, e.g. in products such as diapers and sheets, as well as in the agricultural sector, e.g. in seed pellets.
Decisive factors for the use of the new starch modified by recombinant DNA techniques are, on the one hand, structure, water content, protein content, lipid content, fiber content, ashes/phosphate content, amylose/amylopectin ratio, distribution of the relative molar mass, degree of branching, granule size and shape as well as crystallisation, and on the other hand, the properties resulting in the following features: flow and sorption behavior, pasting temperature, viscosity, viscosity stability in saline solution, thickening performance, solubility, paste structure and transparency, heat, shear and acid resistance, tendency to retrogradation, capability of gel formation, resistance to freezing/thawing, capability of complex formation, iodine binding, film formation, adhesive strength, enzyme stability, digestibility and reactivity.
The production of modified starch by genetically operating with a transgenic plant may modify the properties of the starch obtained from the plant in such a way as to render further modifications by means of chemical or physical methods superfluous. On the other hand, the starches modified by means of recombinant DNA techniques might be subjected to further chemical modification, which will result in further improvement of quality for certain of the above-described fields of application. These chemical modifications are principally known to the person skilled in the art. These are particularly modifications by means of
heat treatment
acid treatment
oxidation and
esterification
leading to the formation of phosphate, nitrate, sulfate, xanthate, acetate and citrate starches. Other organic acids may also be used for the esterification:
formation of starch ethers
starch alkyl ether, O-allyl ether, hydroxylalkyl ether, O-carboxylmethyl ether, N-containing starch ethers, P-containing starch ethers and S-containing starch ethers.
formation of branched starches
formation of starch graft polymers.
In a further embodiment of the invention the foreign nucleic acid molecule has one or more protein targeting signal sequence(s) mediating a cell wall-specific localisation of the amylosucrase protein and the branching enzyme.
The nucleic acid molecules coding for the amylosucrase enzyme and the branching enzyme which are both contained in the xe2x80x9cforeign nucleic acid moleculexe2x80x9d can either be under control of one or of several protein targeting signal sequence(s) independently from each other or they can be under control of one or of several protein targeting signal sequence(s) together after fusion as translational unit.
In another embodiment of the invention the foreign nucleic acid molecules have one or more protein targeting signal sequence(s) each mediating a cell wall-specific localisation of the amylosucrase protein and the branching enzyme protein. In this embodiment several foreign nucleic acid molecules are introduced into the genome of the plant cell wherein one foreign nucleic acid molecule encodes an amylosucrase protein and another foreign nucleic acid molecule encodes a branching enzyme. As mentioned earlier, the foreign nucleic acid molecules can be introduced into the genome of the plant cell simultaneously or consecutively. In the first case it is called xe2x80x9ccotransformationxe2x80x9d, in the latter xe2x80x9csupertransformationxe2x80x9d.
Each of the introduced foreign nucleic acid molecules contains one or more protein targeting signal sequence(s) mediating a cell wall-specific localisation of the amylosucrase protein and the branching enzyme protein each wherein the protein targeting signal sequences are identical or different from each other.
As signal sequence that of the proteinase inhibitor II from potato can be used (von Schaewen et al., EMBO J. 9, (1990), 3033-3044; Keil et al., Nucleic Acid Research 14, (1986), 5641-5650).
In a further embodiment of the invention the foreign nucleic acid molecule(s) mediates (mediate) a cytosolic localisation of the amylosucrase protein and the branching enzyme.
Moreover, the present invention relates to transgenic plants containing such plant cells with increased activity of an amylosucrase and of a branching enzyme.
The plants of the invention can belong to any plant species, i.e. they can be monocotyledonous plants or dicotyledonous plants. Preferably they are plants from agricultural useful plants, i.e. from plants which are cultivated by man for use as foods or for technical, particularly industrial use. The invention preferably relates to fibre-forming plants (e.g. linen, cannabis, cotton), oil-storing plants (e.g rape, sunflower, soybean), starch-storing plants (e.g. wheat, barley, oats, rye, potato, maize, rice, pea, cassava), sugar-storing plants (e.g. sugar beet, sugar cane, sugar millet) and protein-storing plants (e.g. leguminous plants).
In a further preferred embodiment the invention relates to food plants (e.g. forage crop and pasture plants (alfalfa, clover, etc.)), vegetable plants (e.g. tomatoes, salad, chicory). Particularly preferred are sugar beet, sugar cane, maize, wheat and rice.
The present invention also relates to a method for the production of transgenic plants giving an increased yield in comparison with wildtype plants wherein
a) a plant cell is genetically modified by the introduction of a (several) foreign nucleic acid molecule(s) the presence or expression of which leads (lead) to an increased activity of a protein with amylosucrase activity and an increase in the activity of a protein with branching enzyme activity;
b) a plant is regenerated from the cell produced according to a); and
c) further plants are optionally produced from the plant produced according to step b).
Moreover, the present invention relates to a method for the production of a transgenic plant which synthesizes xcex1-1,6 branched xcex1-1,4-glucans with a modified branching a degree in O-6-position in comparison with corresponding genetically non-modified wildtype plants wherein
a) a plant cell is genetically modified by the introduction of one or more foreign nucleic acid molecule(s) the presence or the expression of which leads (lead) to an increased activity of a protein with the activity of an amylosucrase and an increased activity of a protein with the activity of a branching enzyme;
b) a plant is regenerated from the cell produced according to a); and
c) further plants are optionally produced from the plant produced according to step b).
Another subject-matter of the present invention is a method for the production of a transgenic plant synthesizing a modified starch in comparison with corresponding genetically non-modified wildtype plants wherein
a) a plant cell is genetically modified by the introduction of one or more foreign nucleic acid molecule(s) the presence or the expression of which leads (lead) to an increased activity of a protein with the activity of an amylosucrase and an increased activity of a protein with the activity of a branching enzyme;
b) a plant is regenerated from the cell produced according to a); and
c) further plants are optionally produced from the plant produced according to step b).
The same as described above in another context concerning the plants of the invention applies to the genetic modification introduced according to step a).
The regeneration of plants according to step b) can be carried out according to methods known to the person skilled in the art.
The generation of further plants according to step c) of the method of the invention be achieved e.g. through vegetative propagation (for example via cuttings, tubers or via callus culture and regeneration of whole plants) or through sexual reproduction. The sexual reproduction is preferably carried out under control, i.e. selected plants with certain properties are crossed with each other and propagated.
The present invention also relates to the plants obtainable by the methods of the invention.
The person skilled in the art knows that he can obtain the plants of the invention not only through the aforementioned methods of the invention but also by crossing, for example, a genetically modified plant which has an increased activity of a protein with amylosucrase activity due to the introduction of a foreign nucleic acid molecule with a transgenic plant which has an increased activity of a protein with branching enzyme activity due to the introduction of a foreign nucleic acid molecule. Furthermore it is known to the person skilled in the art that the supertransformation described above is not by all means to be carried out with primary transformants but preferably with stable transgenic plants which have been selected before and which, favourably, have been tested in corresponding experiments with regard to, for example, fertility, stable expression of the foreign gene, hemi- and homozygosity etc. Therefore, also tansgenic plant cells and plants are subject-matter of the present invention which are obtainable by the aforementioned methods and which show the phenotype described in the embodiments above.
The present invention also relates to propagation material of the plants of the invention as well as of transgenic plants produced according the methods of the invention. The term xe2x80x9cpropagation materialxe2x80x9d comprises those components of the plant which are suitable for the production of descendants in a vegetative or generative way. For the vegetative propagation, for example, cuttings, callus cultures, rhizomes or tubers are suitable. Other propagation material comprises, for example, fruit, seeds, seedlings, protoplasts, cell cultures etc. Preferably, the propagation material are tubers and seeds.
Furthermore, the present invention relates to the use of one or more nucleic acid molecule(s) encoding a protein with the enzymatic activity of an amylosucrase and a protein with the enzymatic activity of a branching enzyme for the production of plants which give an increased yield in comparison with wildtype plants and/or synthesize starch which is modified in comparison with starch from wildtype plants and/or synthesize xcex1-1,6 branched xcex1-1,4-glucans with a modified branching degree in O-6 position in comparison with corresponding genetically non-modified wildtype plants.